The agglutination reaction has long been used in visual (semiquantitative) and quantitative assays for a wide variety of bacteria, cell-surface antigens, serum proteins or other analytes of clinical interest. Agglutination results from the reaction of bivalent antibodies with multivalent antigens of interest to produce aggregates which can be detected and/or measured in various ways. Similarly, the same reaction can be utilized for the detection of specific antibodies by the agglutination reaction caused by the addition of the corresponding antigen.
In order to produce large, crosslinked aggregates the number of reactive sites on the antigens must be at least 2. Therefore, when the detection of monovalent haptens was desired, the reaction scheme was modified as follows: A multivalent form of the antigen such as a hapten-protein conjugate was prepared and the hapten present in a sample could compete with its multivalent form for the available binding sites of the antibody thereby reducing the amount of agglutination. This technique is referred to as inhibition of agglutination.
Production of multivalent forms of haptens is old in the art. Frequently the hapten is bonded to a carrier protein as is done in the preparation of immunogens. The stoichiometry of the reaction can be adjusted to place three or more haptens per protein molecule, the exact number being determined by the needs of the particular assay in which the material will be utilized.
Increased sensitivity to visual or instrumental detection of agglutination or its inhibition can be achieved by the use of particle reagents as carriers, rather than soluble proteins or protein conjugates. It has been shown, for example, that antiserum to hen ovalbumin was 2000-fold more sensitive in precipitating hen ovalbumin coated on colloidion particles than in precipitating hen albumin itself; H. N. Eisen, "Immunology", Harper and Row, 1974, page 394.
Antibody particle reagents are also known. A common method for preparation of such reagents is by adsorption of the antibodies onto the surface of suitable adsorbents. Polystyrene-based latex particles have been used extensively for this purpose. For example, U.S. Pat. No. 3,912,805, issued Oct. 14, 1975 to Cayzer, et al., describes an immunological diagnostic reagent wherein antibodies raised to fibrinogen degradation products are adsorbed to latex particles. These reagents, however, are susceptible to desorption during storage or use leading to variations in reagent properties. This, in turn, can adversely affect assay sensitivity and reproducibility.
To overcome the problems of desorption, particle reagents can be prepared by covalent attachment of the compounds of biological interest to the surface of particles. Polystyrene polymers have been modified to include functional groups capable of covalent protein attachment. U.S. Pat. No. 4,064,080, issued Dec. 20, 1977, discloses styrene polymers with terminal aminophenyl groups and proteins attached to them. U.S. Pat. No. 4,181,636, issued Jan. 1, 1980, discloses carboxylated latex polymers coupled to immunologically active materials through a water soluble activating agent and their use as diagnostic reagents in agglutination tests. U.S. Pat. No. 4,210,723, issued July 1, 1980, describes shell-core latex polymer particles of 0.15-1.5 .mu.m diameter having free epoxy groups on the surface of the particles and the coupling of proteins through these epoxy groups.
Other polymeric systems have also been developed for later attachment of immunologically active materials. U.S. Pat. No. 4,264,766, issued Apr. 28, 1981, discloses latex polymers, having a particle size of 0.01-0.9 .mu.m and having active groups such as carboxyl and amino groups to which water soluble polyhydroxy compounds can be attached covalently. Through the utilization of activating agents such as carbodiimides, immunologically active materials were attached to the latex particle/polyhydroxy compound carriers to form diagnostically useful reagents.
There is a need for a stable particle reagent which possesses high sensitivity for use in light-scattering agglutination assays and which can be prepared conveniently by the covalent attachment of compounds of biological interest to a particulate carrier.